Glycine max (L.) Merr. (soybean) is the second largest crop in the United Sates, with an estimated annual value of 11 billion dollars. Plant parasites such as the Heterodera glycines Ichinohe and Meloidogyne spp. cause significant damage to soybean, with diseased plants exhibiting symptoms ranging from stunting, chlorosis and wilting to enhanced susceptibility to other diseases. Recent estimates of annual production losses by the H. glycines, the most damaging soybean pest, range from $460 million to 818 million for the US alone (Wrather and Koenning 2006). Nematicides, crop rotation and resistant varieties represent the current options for H. glycines management; however, each has serious limitations. Nematicides, including organophosphate and carbamate compounds, are extremely toxic and increase production costs. Crop rotation can require prolonged intervals without a host crop to be effective. Resistant cultivars have a narrow genetic base, while H. glycines populations display broad genetic diversity leading to frequent virulence selection (Dong et al. 1997). Many populations of H. glycines, for instance, are now able to reproduce on soybean cultivars derived from PI88788, the most widely used source of H. glycines resistance in the USA (Mitchum et al. 2007; Hershman et al. 2008). It is therefore imperative that new strategies for H. glycines control be explored to complement existing approaches.
Genetic engineering represents one promising approach to H. glycines management, but improving nematode resistance in plants through this method requires increased knowledge of potential target genes. The search for novel targets for genetically engineered resistance to H. glycines has led to intense study of the secretions of subventral and dorsal esophageal gland cells of the nematode, as they play important roles in the host-parasite interaction. As a result of these studies, genes encoding secreted proteins of H. glycines have been identified, including genes encoding polygalacturonase (Mahalingam et al. 1999) and chorismate mutase (Bekal et al. 2003). Numerous additional putative H. glycines parasitism genes have been identified using microarray analysis (Klink et al. 2007; Ithal et al. 2007; Klink et al. 2009a, b). Although the functions of many of these genes remain to be investigated, Alkharouf et al. (2007) and Klink et al. (2009c) have identified specific genes involved in female development by knocking out these genes' functions in vitro and in vivo, respectively.
RNA interference (RNAi) is a potentially powerful gene-silencing tool for analysis of gene function. The mechanism of RNAi was first identified in the free-living nematode Caenorhabditis elegans, in which the expression of unc22 gene was suppressed via the RNAi pathway (Fire et al. 1998). During this process, long double-stranded RNA is processed into 21-23 nucleotide siRNAs by Dicer, a member of the RNase family (Bernstein et al. 2001). The DCR-2/R2D2 complex binds to siRNAs and enhances sequence-specific messenger RNA degradation mediated by the RNA-initiated silencing complex (Liu et al. 2003). This pathway recently has shown promise as the basis of a novel control strategy for plant-parasitic nematodes, with numerous independent studies demonstrating suppression of target nematode populations following soaking nematodes in dsRNA solutions (Urwin et al. 2002; Bakhetia et al. 2005; Huang et al. 2006; Alkharouf et al. 2007) and, more importantly, using in planta transgenic systems expressing dsRNA fragments of nematode genes (Huang et al. 2006; Steeves et al. 2006; Yadav et al. 2006; Sindhu et al. 2009). Yadav et al. (2006) reported that RNAi was induced by using dsRNA fragments of two genes encoding an integrase and a splicing factor in the plant-parasitic nematode M. incognita, leading to protection against nematode infection in tobacco. The expression of root-knot nematode parasitism gene 16D10 dsRNA in transgenic Arabidopsis resulted in resistance against four major root knot nematode species (Huang et al. 2006), while Sindhu et al. (2009) obtained reductions in H. schachtii females ranging from 23 to 64% in transgenic Arabidopsis lines expressing RNAi constructs of four parasitism genes. RNA interference appears to be similarly effective against H. glycines in transformed soybean lines. Steeves et al. (2006) successfully produced transgenic soybean lines using this RNAi strategy targeting a major sperm protein of H. glycines. Bioassay data indicated transgenic plants had up to a 68% reduction in eggs g−1 root tissue. The effects of plant-derived dsRNA molecules appeared to continue into the next generation.
Targets for host-delivered RNAi suppression of plant parasitic nematodes can be selected based on known RNAi effects on corresponding C. elegans genes. Alkharouf et al. (2007), for instance, used bioinformatics to yield 1,508 candidate H. glycines genes whose homologous genes of C. elegans have lethal phenotypes when silenced in C. elegans. They also reported in vitro silencing a conserved ribosomal gene from H. glycines (Hg-rps-23) resulted in dead and dying worms as shown by positive Sytox fluorescence. Klink et al. (2009c) used microarray analysis to demonstrate that 32 of 150 conserved H. glycines homologues of C. elegans genes with lethal phenotypes were induced during feeding site establishment, and subsequently inhibited female development by engineering transgenic soybean plants with tandem inverted repeats of selected homologs.
Novel approaches to SCN management are needed to complement current strategies, and prolong the effectiveness of available resistance genes.